The active metabolites of the B vitamin folic acid are essential cofactors for many biochemical reactions involving one-carbon transfers. Folate deficiency has been associated with an increased incidence of several forms of cancer and recent studies with the human colon cancer cell lines suggest that only certain forms of folate might inhibit colon cancer cell proliferation. In addition, maternal folate has been shown to prevent neural tube defects such as spina bifida and anencephalus. Since folic acid fortification of enriched cereal was initiated in the US, the incidence of spina bifida has declined 20 percent, but the incidence of anencephaly remains unchanged, which supports the need for alternate types or higher levels of dietary folates. Since folic acid must be metabolized to its reduced forms for biological activity, perhaps one of these pre-formed active metabolites of folic acid would be more effective for the prevention of cancer and birth defects. Furthermore, a greater understanding of the bioavailability of dietary folates including polyglutamyl forms of folate is essential for establishing dietary guidelines for specific population groups and for making accurate decisions with respect to food fortification. To address these issues, new highly sensitive and selective analytical methods are needed to simultaneously measure multiple forms of folates in blood and cells. We have reported a new HPLC-tandem mass spectrometry (LC-MS-MS) assay based on hydrophilic interaction chromatography coupled with negative ion tandem mass spectrometry for the analysis of 5'-methyl-tetrahydrofolate in human plasma. As Specific Aim 1 of our investigation, we propose to expand this assay to include the simultaneous measurement of multiple forms of folate including folic acid, tetrahydrofolate (THF), 5-methyl-THF, and 5'-formyl THF in human plasma and human cells grown in culture. To the best of our knowledge, no other laboratory has reported the measurement of all of these folates in human plasma or tissues. Then as Specific Aim 2, we will apply our new LC-MS-MS assay to the quantitative analysis of multiple forms of labeled and unlabeled folates in human plasma in support of an on-going clinical study of the bioavailability of intrinsically labeled [13C11]-folic acid and [13C6]-hexaglutamyl folic acid. These studies will open a wide range of clinical and basic science research opportunities for nutrition-based cancer chemoprevention, which will become the basis of subsequent R01-type grant applications. Finally as Specific Aim 3, the transport and metabolism of labeled folic acid, hexaglutamyl folic acid, 5-methyl-THF, 5-formyl-THF, and THF will be investigated using Caco-2, which form a highly differentiated monolayer in cell culture that is a standard model for the human instestinal uptake of orally administered compounds. Our new LC-MS-MS assay and the use of 13C-labeled folate species will provide new and more detailed information on the uptake, metabolism, and bioavailability of folates than has been possible previously.